Gene encoding a novel extracellular metalloprotease in Bacillus subtilis

Abstract

The gene for a novel extracellular metalioprotease was cloned, and its nucleotide sequence was determined. The gene (mpr) encodes a primary product of 313 amino acids that has little similarity to other known Bacillus proteases. The amnmo acid sequence of the mature protease was preceded by a signal sequence of approximately 34 amino acids and a pro sequence of 58 amino acids. Four cysteine residues were found in the deduced amino acid sequence of the mature protein, indicating the possible presence of disulfide bonds. The mpr gene mapped in the cysA-arol region of the chromosome and was not required for growth or sporulation.The gram-positive, spore-forming bacterium Bacillus subtilis produces and secretes proteases, esterases, and other types of exoenzymes at the end of the exponential phase of growth (15). The principal extracellular proteolytic enzymes, alkaline (subtilisin) and neutral (metallo-) proteases, are encoded by the apr and npr genes, respectively (11,24,26,29). In addition, the genes for two minor extracellular proteases, epr (21) and bpr (encoding bacillopeptidase F; A. Sloma, G. A. Rufo, Jr., C. F. Rudolph, B. J. Sullivan, K. A. Theriault, and J. Pero, submitted for publication) have been identified. Strains bearing null mutations in these four protease genes and the major intracellular protease gene isp-i (12) still produce extracellular protease activity. The majority of the residual protease activity can be attributed to a novel cysteine-containing metalloprotease (Mpr) that has been purified to apparent homogeneity (17). Here we report the cloning of the gene encoding this cysteine-containing protease. MATERIALS AND METHODSBacterial strains and plasmids. B. subtilis strains are listed in Table 1. Plasmid pBs81/6 is a derivative of pBD64 (8). Plasmids pBR322 and pUC19 were used for cloning into Escherichia coli DH5 cells obtained from Bethesda Research Laboratories, Inc., Gaithersburg, Md. The cat gene was obtained from plasmid pMI1101 (30), and the ble gene was obtained from pUB110 (20). B. subtilis strains were grown on tryptose blood agar base or minimal glucose medium and were made competent by the procedure of Anagnostopoulos and Spizizen (1). Selection for phleomycin resistance was carried out on tryptose blood agar base by the overlayer method after a 1.5-h delay at 37°C to allow for expression.The final concentration of phleomycin was 2 jig/ml. Plasmid DNA from B. subtilis and E. coli was prepared by the alkaline lysis method of Birnboim and Doly (3). Plasmid DNA transformation in B. subtilis was performed as described by Gryczan et al. Amino acid sequence determination. The N-terminal amino acid sequence of purified Mpr was determined by automated sequential Edman degradation with subsequent identification and quantification of phenylthiohydantoin-labeled amino acids by reverse-phase high-performance liquid chromatography. Additional amino acid sequences were obtained from internal fragments of Mpr generated by trypsin digestion. The purified enzyme was incubated with trypsin, and t...

Cloning and sequencing of a gene encoding a novel extracellular neutral proteinase from Streptomyces sp. strain C5 and expression of the gene in Streptomyces lividans 1326

“…Finally, the similarity in sizes of the SnpAs from S. galilaeus(pANT42) and S. lividans(pANT42) also indicates that these two organisms recognize and cleave off the leader peptide from mature SnpA in an analogous manner. The 81 amino acids upstream of the N terminus of the mature proteinase probably constitute a leader sequence, as has been found with most bacterial extracellular proteinases (11,22,37,49,56,60). The N terminus of this region, however, is somewhat different from most bacterial leader sequences (58) in that it contains only a single positively charged amino acid (Arg) that is placed between the two Met residues.…”

“…No sequences that contained significant homology with SnpA outside of the conserved zinc ligand site were found in the Genbank or EMBL data base (dated June 1991). Similarly, the deduced amino acid sequence of SnpA was compared directly (one-to-one) with the amino acid sequences of Bacillus thermoproteolyticus thermolysin (54), carboxypeptidase A (7), Pseudomonas aeruginosa elastase (3), S. cacaoi metalloproteinase (11), two Bacillus subtilis metalloproteinases (49,60), and proteinase B from Erwinia chrysanthemi (13); no significant homology was found between SnpA and any of these other metalloproteinases. This is not necessarily surprising, given the differences between SnpA and these other neutral proteinases with respect to size, substrate specificity, and sensitivity to inhibitors.…”

Generation of Recombinant Human Ache Op-Scavengers With Extended Circulatory Longevity

“…For instance, proteases of various species of Bacillus may severely affect production and secretion of foreign proteins by these bacteria. To remedy this situation, mutant strains that produce less proteases were generated (Sloma et al, 1990;Wu et al, 1991; Ye ef al., 1999;Lee et al, 2000).…”

“…For instance, proteases of various species of Bacillus may severely affect production and secretion of foreign proteins by these bacteria. To remedy this situation, mutant strains that produce less proteases were generated (Sloma et al, 1990;Wu et al, 1991; Ye ef al., 1999;Lee et al, 2000).Progressive mutagenesis of Bacillus suhtilis resulted in the generation of strains which are deficient in two (subtilisin and neutral protease, the major B. subtilis proteases), five, six and seven proteases. The latter strain, WB700, allowed 8-fold higher production/secretion of staphylokinase, than wild type B. subtilis (~340mg/ml under optimized conditions.…”

Molecular cloning and nucleotide sequence of the gene encoding a calcium-dependent exoproteinase from Bacillus megaterium ATCC 14581

“…In the past, many protease genes from representatives of the genus have been cloned and characterized. For example, Bacillus subtilis secretes many different extracellular proteases into the culture medium, especially at the end of the exponential growth phase (Stahl & Ferrari, 1984;Wong et al, 1984;Yang et al, 1984;Sloma et al, 1990;Wu et al, 1990;Tran et a/., 199 1). The neutral proteases from B. stearothermophilus (Fuji et al, 1983;Takagi et a/., 1985;Nishiya & Imanaka, 1990), B. thermoproteolyticus (thermolysin) (Latt et a/., 1969 ;Staufer, 1971 ;Titani et a/., 1972;Matthews et al, 1972), B. cereus (Sidler et al, 1986;Pauptit et a/., 1988), and The sequence data reported in this paper have been submitted to Gen Bank and have been assigned the accession number X61380 NPRM.…”